Abstract: Historically, most research on cell fate induction was focused around biochemical signals. It is now, however, clear that mechanical signalling from the extracellular matrix (ECM) also influences fate in various cell types. For example, in human pluripotent stem cells (hPSCs), softening of the substrate increases mesoderm differentiation. Although such phenomena have been observed in different systems, the mechanisms underlying the effect of mechanical properties of the ECM on stem cell fate remain largely unstudied. Here, we investigate such mechanism in hPSCs. We cultured hPSCs on stiff and soft polyacrylamide hydrogels and exposed the cells to the mesoderm-inducing BMP4 signal. On the stiff substrate, hPSCs display BMP activity around the edge of their colony, however, on the soft substrate, they respond within the whole colony and consequently exhibit increased differentiation. We show that the increased response to BMP on the soft substrate stems from the lower activity of the Focal Adhesion Kinase (FAK)-PI3K cascade. The lower FAK-PI3K activity results in morphological changes of the hPSC epithelium. Specifically, substrate softening affects two crucial epithelial properties in hPSCs: epithelial integrity and epithelial polarity. hPSCs on the soft substrate exhibit increased epithelial permeability and loss of apical membrane identity. Both of these changes allow increased accessibility of the BMP receptor to the BMP ligand on the soft substrate, allowing in turn the observed increase in BMP signalling activity. Finally, we show that these changes in epithelial properties on the soft substrate stem from increased retention of endogenous laminin on the apical surface of hPSCs. Together, our work identifies a mechanism through which a change in substrate stiffness affects the response of hPSCs to BMP and the subsequent extent of differentiation. We are currently investigating whether such mechanism is involved in initiation of gastrulation in the epiblast since remodelling of the underlying basement membrane is necessary for the start of gastrulation. More broadly, our findings should impact our understanding of how physiological changes in ECM mechanics impact cellular response to biochemical signals and consequent fate/state transitions during processes like differentiation or metastasis.
Funding Source: This research has been funded by the Wellcome Trust.
